The present invention relates to a promoter and to a construct comprising the same.
In particular, the present invention relates to a chemically inducible gene promoter sequence, and particularly, but not exclusively, a chemically inducible gene promoter sequence based on cis regulatory elements from the maize glutathione S-transferase 27 (GST-27) gene. The present invention also relates to gene constructs, expression systems, plants and promoter/inducer combinations comprising the chemically inducible gene promoter sequence.
Recent advances in molecular biology techniques have resulted in a better understanding of plant promoters. Cis-regulatory elements have been identified and used to localise reporter gene activity to specific differentiated cell types and to defined stages of plant development (Drews et al., 1992; Guerrero et al., 1990). While current technology exists to regulate trans-gene activity in a spatial or temporal manner, the external control of introduced genes by application of an inducing chemical is not well established in plants.
The ability to regulate genes in an inducible manner is well established in bacteria, fungi, insects and animal cell cultures. For inducible regulation systems to be effective there should be a zero or low level of expression in the absence of inducer, high expression following treatment with inducer and no effect of the inducer on other cellular functions.
Whilst numerous inducible genes have been isolated from plants (Kuhlemeier et al., 1987), a well defined inducible regulation system is not in common use. A number of genes have been described which are activated by pathogen attack or environmental stimuli, including light, oxygen and temperature levels. Although some of these are well characterised at the molecular level, they cannot be utilised for inducible genes system due to illegitimate activation by environmental signals.
The involvement of chemical stimuli, including plant growth regulators, in activation of gene transcription is well documented in plants. Application of these compounds may be better controlled, in comparison with environmental stimuli, however they cannot be considered for inducible genes systems due to undesirable pleiotropic effects.
A number of recent studies have demonstrated that control of trans-genes in plants can be achieved by application of exogenous chemicals. These include activation by salicylic acid (Williams et al., 1992), tetracyline (Weinmann et al., 1994], glucocorticoids (Schena et al., 1991) and copper ions (Mett et al., 1993). Although these systems fulfil the prerequisites described earlier, and therefore have utility for research applications, their use will be limited as the chemicals described are not compatible with current agricultural practice.
A potentially attractive group of chemicals which may have utility in regulating gene expression in transgenic plants are herbicide safeners. These compounds are currently used in agriculture and function to selectively elevate the metabolism of certain herbicides, primarily by inducing the detoxifying enzymes, glutathione S-transferase (Hatzios, 1991) and cytochrome p450-dependent mixed function oxidises (Fonne-Pfister and Kreuz, 1990). Glutathione S-transferases (GSTs) are multi-functional enzymes which catalyze the conjugation of the thiol group of glutathione to electrophilic centres of lipophilic compounds leading to their detoxification (Mannervik and Danielson, 1988). GSTs are ubiquitous and their role in xenobiotic metabolism in mammals and plants (Lamoureux and Rusness 1989) is well established.
The best characterised system of plant GSTs is found in maize, where they account for 1-2% of soluble protein (Timmerman, 1989). Four isoforms of GST have been described in maize, GST I (Mozer et al., 1983; Weigand et al., 1986), GST II (Mozer et al., 1983; Holt et al., 1995), GST III (Moore et al., 1986) and GST IV (Irzyk and Feurst, 1993). GST-27 is a component of GST II and GST IV which exhibit safener dependent inducibility.
In our International Patent Publication No. W093/01294, the teaching of which is hereby incorporated by reference, we demonstrated that the promoter region controlling GST-27 can be used to achieve safener dependant trans-gene expression. These studies revealed that a 3.8 kb GST-27 promoter, in addition to directing safener inducible trans-gene expression, also gave a constitutive level of expression in root tissues.